MICRORNA INHIBITORS FOR USE IN TREATING METABOLIC DISEASES

Abstract

The present invention relates to composition comprising an inhibitor of miR-379 or a portion or fragment thereof and an inhibitor of miR-541 or a portion or fragment thereof, and/or an inhibitor of the target site of miR-379 or a portion or fragment thereof and an inhibitor of the target site of miR-541 or a portion or fragment thereof, and/or a combination of an inhibitor of miR-379 or a portion or fragment thereof and an inhibitor of the target site of miR-541 or a portion or fragment thereof or a combination of an inhibitor of the target site of miR-379 or a portion or fragment thereof and an inhibitor of miR-541 or a portion or fragment thereof. The present invention also relates to the respective composition for use in treating or preventing a metabolic disease, a disease related to a metabolic disorder, and/or cancer.

Claims

1. A composition comprising (a) an inhibitor of miR-379 or a portion or fragment thereof and an inhibitor of miR-541 or a portion or fragment thereof, and/or (b) an inhibitor of the target site of miR-379 or a portion or fragment thereof and an inhibitor of the target site of miR-541 or a portion or fragment thereof, and/or (c) a combination of an inhibitor of miR-379 or a portion or fragment thereof and an inhibitor of the target site of miR-541 or a portion or fragment thereof or a combination of an inhibitor of the target site of miR-379 or a portion or fragment thereof and an inhibitor of miR-541 or a portion or fragment thereof.

2. The composition of claim 1, wherein at least one inhibitor of miR-379 or a portion or fragment thereof comprises a nucleotide sequence complementary to or hybridizing to miR-379 or a portion or fragment thereof, and at least one inhibitor of miR-541 or a portion or fragment thereof comprises a nucleotide sequence complementary to or hybridizing to miR-541 or a portion or fragment thereof.

3. The composition of claim 1, wherein a portion of miR-379 has a nucleotide sequence according to SEQ ID NO: 1, wherein not more than 6 nucleotides are substituted, and a portion of miR-541 has a nucleotide sequence according to SEQ ID NO: 2, wherein not more than 8 nucleotides are substituted.

4. The composition of claim 1, wherein at least one inhibitor comprises a nucleic acid sequence comprising at least 10 nucleotides.

5. The composition of claim 1, wherein at least one inhibitor is selected from the group consisting of Tough Decoys (TuD), Decoys, antisense oligonucleotides, anti-miR, block-miR, ribozymes, external guide sequence (EGS), oligonucleotides, small interference RNA (siRNA), small temporal RNA (stRNA), short hairpin RNA (shRNA), small RNA-induced gene activation (RNAa), small activating RNA (saRNA), locked nucleic acids (LNA), antagomirs, and peptide nucleic acids (PNA).

6. The composition of claim 5, wherein at least one inhibitor is a Tough Decoy RNA (TuD).

7. The composition of claim 1, wherein at least one inhibitor comprises a chemical modification of the nucleic acid sequence selected from the group consisting of nucleic acid analogs comprising N-acetylgalactosamine (GalNAc), phosphorothioate DNA (PS), 2′-O-methyl RNA (OMe), 2′-O-methoxy-ethyl RNA (MOE), peptide nucleic acid (PNA), N3′-P5′-phosphoroamidate (NP), 2′-fluoro-arabino nucleic acid (FANA), morpholino phosphoroamidate (MF), cyclohexene nucleic acid (CeNA), and tricycleDNA (tc-DNA).

8. The composition of claim 1, wherein at least one inhibitor is comprised by a delivery vehicle selected from the group consisting of adeno-associated virus (AAV), lentiviral vector, polyethylene imine (PEI), cationic liposomes, silica nanoparticles, PEGylated PLGA, and neutral lipid.

9. The composition of claim 1, wherein at least one inhibitor is comprised by an adeno-associated virus (AAV).

10. The composition of claim 1, wherein the inhibitor of miR-379 or a portion or fragment thereof comprises a nucleotide sequence of SEQ ID NO: 3, wherein not more than 5 nucleotides are substituted, and/or the inhibitor of miR-541 or a portion or fragment thereof comprises a nucleotide sequence of SEQ ID NO: 4, wherein not more than 5 nucleotides are substituted.

11. The composition of claim 1, comprising an inhibitor of miR-379 or a portion or fragment thereof and an inhibitor of miR-541 or a portion or fragment thereof on the same molecule, wherein said molecule comprises a nucleotide sequence complementary to or hybridizing to miR-379 or a portion or fragment thereof and a nucleotide sequence complementary to or hybridizing to miR-541 or a portion or fragment thereof.

12. The composition of claim 11, wherein said inhibitor comprises the nucleotide sequence of SEQ ID NO: 5, wherein not more than 10 nucleotides are substituted.

13. The composition of claim 1, which is a pharmaceutical composition.

14. The composition of claim 1 for use in treating or preventing a metabolic disease, a disease related to a metabolic disorder, and/or cancer.

15. The composition of claim 14, wherein said metabolic disease or disease related to a metabolic disorder is selected from the group consisting of glucocorticoid hormone driven metabolic dysfunction, obesity, diabetes, diabesity, metabolic syndrome, insulin resistance, hyperglycemia, (systemic) dyslipidemia, Cushing's syndrome, adverse or side effects associated with or caused by glucocorticoid (GC) treatment or excess, atherosclerosis, heart disease, stroke, (cancer) cachexia, growth defects, hepatic steatosis, NASH, and liver fibrosis.

Description

[0047] The Figures show:

[0048] FIG. 1: Experimental protocol for the liver-specific inhibition of miR-541 and miR-379 activity by rAAV-mediated delivery of TuD inhibitors in mice.

[0049] FIG. 2: Reduction in HOMA-IR (surrogate measurement of insulin resistance) observed in animals with hepatic-specific inhibition of miR-541 and miR-379 activity (AAV-TuD, white bar) as compared to negative control (AAV-NC, black bar).

[0050] FIG. 3: Decreased circulating levels of triglycerides in animals with hepatic-specific inhibition of miR-541 and miR-379 activity (AAV-TuD, white bar) as compared to negative control (AAV-NC, black bar).

[0051] FIG. 4: Improvement in glucose clearance following an intraperitoneal glucose load (2 g/kg) in mice with hepatic-specific inhibition of miR-541 and miR-379 activity (AAV-TuD, open circles and white bar) as compared to negative control (AAV-NC, closed squares and black bar). Glucose profile (A) and area under the curve (B).

[0052] FIG. 5: Improvement in glucose tolerance following an intraperitoneal glucose load (ipGTT, 2 g glucose/kg) performed on week 4 of therapy in wild-type mice with combined hepatic-specific inhibition of miR-541 and miR-379 (FIG. 5A, same profile as presented in FIG. 4), hepatic-specific inhibition of miR-379 (FIG. 5B) or hepatic-specific inhibition of miR-541 activity (FIG. 5C) (AAV-TuD, open circles in the upper graph and white bars in the lower graph of each panel) as compared to negative control (AAV-NC, closed squares in the upper part and black bars in the lower part of each panel). The time-course glucose profile is presented in the upper graph, while the corresponding area under the curve is shown in the lower graph of each panel. A synergistic improvement in glucose clearance was observed in response to the combined inhibition of miR-541 and miR-379 activity.

[0053] FIG. 6: Blood glucose levels in response to an exogenous insulin bolus (0.7 IU insulin/kg) intraperitoneally administered on week 3 of therapy to wild-type mice with combined hepatic-specific inhibition of miR-541 and miR-379 (FIG. 6A), hepatic-specific inhibition of miR-379 (FIG. 6B) or hepatic-specific inhibition of miR-541 activity (FIG. 6C) (AAV-TuD, open circles in each FIG. 6A-C) as compared to negative control (AAV-NC, closed squares in each FIG. 6A-C). Only in animals carrying hepatic-specific inhibition of both miR-541 and miR-379 activity, a significant reduction in glucose levels was found at all time points studied, as compared to negative control animals.

[0054] FIG. 7: Plasma triglyceride levels (5-6 h fasting) 2, 3 and 4 weeks after treatment in wild-type mice with combined hepatic-specific inhibition of miR-541 and miR-379 (FIG. 7A), hepatic-specific inhibition of miR-379 (FIG. 7B) and hepatic-specific inhibition of miR-541 activity (FIG. 7C) (AAV-TuD, white bars) as compared to negative control (AAV-NC, black bars). A robust lowering effect of circulating triglycerides was observed in response to the combined hepatic-specific inhibition of miR-541 and miR-379 activity.

[0055] The following sequences are provided herein:

TABLE-US-00001 RNA H. sapiens miR-379-5p bold: complementary sequence to inhibitor SEQ ID NO: 3 SEQ ID NO: 1 5′-UGGUAGACUAUGGAACGUAGG-3′ RNA H. sapiens miR-541-5p bold: complementary sequence to inhibitor SEQ ID NO: 4 SEQ ID NO: 2 5′-AAAGGAUUCUGCUGUCGGUCCCACU-3′ DNA artificial Inhibitor of miR-379 SEQ ID NO: 3 5′-GTTCCATAGTCTACC-3′ DNA artificial Inhibitor of miR-541 SEQ ID NO: 4 5′-CGACAGCAGAATCCTT-3′ RNA artificial Inhibitory sequence to miR-379 and miR-541 comprised by TuD SEQ ID NO: 5 5′-GACGGCGCUA GGAUCAUCAA CAGUGGGACC GACAGCAUCU AGAAUCCUUU CAAGUAUUCU GGUCACAGAA UACAACCCUA CGUUCCAAUC UUAGUCUACC ACAAGAUGAU CCUAGCGCCGUC-3′

[0056] The invention is further illustrated by the following examples, however, without being limited to the example or by any specific embodiment of the examples.

EXAMPLES

[0057] Levels of expression of different microRNAs belonging to the Dlk1-Dio3 locus were determined by semiquantitative real-time PCR using TaqMan microRNA assays in liver biopsies from healthy volunteers (n=10) and obese subjects (n=37) who were not on diabetes medications. A consistent upregulation of the microRNAs examined (miR-127, miR-337, miR-379, miR-382, miR-134, miR-541, miR-409) was observed in the liver samples from obese subjects. The two microRNAs showing the highest increments in their expression were miR-379 and miR-541. Significant correlations between the levels of expression of these transcripts and different metabolic indicators were detected, as shown for miR-541 in Table 1.

[0058] The impact of hepatic miR-541 and miR-379 activity inhibition for metabolism was studied in vivo by the rAAV-delivery of tough decoy (TuD) inhibitors under the control of the hepatic-specific LP1 promoter. Generation of the construct delivered by the AAVs were carried out according to Rose A J et al. Cell Metab 2011, 14(1): 123-30. Briefly, to clone these inhibitors into the construct delivered by the rAAV vector, the negative control sequence from the original vector was replaced by the tough decoy sequence using BglII and SalI restriction enzymes. These inhibitor types were previously demonstrated to strongly inhibit the activity of their target microRNAs in vitro (unpublished observations). In three separate studies of the inventors of the present invention, C57BL/6J mice (12 animals per group) were administered with AAVs (5×10.sup.11 viral genomes per mouse) expressing a negative control sequence or the tough decoy inhibitor against both miR-541 and miR-379 (study 1, sequence according to SEQ ID NO: 5), a negative control sequence or the tough decoy inhibitor against miR-379 (study 2), and a negative control sequence or the tough decoy inhibitor against miR-541 (study 3). Body weight as well as food and water intake were monitored regularly, ipGTTs (2 g glucose/kg) were conducted 2 and 4 weeks after virus administration, while an ITT (0.7 IU/kg) was performed 3 weeks after the onset of the study, in both cases after fasting the animals for 6 h prior to the tests, which started between 14:00-15:00 h (a schematic representation of the experimental protocol is depicted in FIG. 1). In addition, postprandial blood samples were collected at 23:00 h on weeks 2.5 and 4.5. The experiments were terminated 5 weeks after the administration of the viral vectors, half of the animals (n=6 mice per group) were killed at 14:00 h after a 5-6 h fasting, while the other half were killed at 23:00 h during the postprandial state. In study 1, no differences on body weight, food or water intake were detected between the animals receiving the negative control sequence and the animals carrying the combined hepatic-specific inhibition of miR-541 and miR-379 activity (AAV-TuD). Fasting glucose was significantly lower in the AAV-TuD group from week 2 till the end of the study, while fasting insulin concentrations were significantly reduced from week 3, and hepatic insulin resistance, as estimated by the homeostatic model assessment (HOMA-IR) index, which is calculated from fasting plasma insulin (FPI) and fasting plasma glucose (FPG) concentrations [FPI (mU/I)×FPG (mmol/l)/22.5], was found to be significantly lower in the AAV-TuD group from week 2 till termination (FIG. 2). In addition, plasma triglyceride levels were also significantly reduced in this group irrespectively of the feeding conditions of the animals (FIGS. 3 and 7A). Glucose clearance was also significantly better in the AAV-TuD group (60% improvement in week 4, p<0.001; FIGS. 4A, 4B and 5A). Despite a significant improvement in glucose clearance was also observed in the animals receiving the other two tough decoy inhibitors tested, the effect was less pronounced (14% and 36% improvement in response to the single tough decoys against miR-379, FIG. 5B, and miR-541 activity, FIG. 5C, respectively), indicating a synergistic effect in response to the simultaneous inhibition of both microRNAs. Moreover, the combined inhibition of both microRNAs also induced a remarkable potentiation of the glucose-lowering effect in response to an exogenous bolus of insulin, with significantly (p<0.001) reduced glucose levels as compared to negative control that were sustained over a two hour period (FIG. 6A). Again, this effect was not matched by the single inhibition of either of the two microRNAs (FIGS. 6B and 6C). Blood glucose levels were determined by glucose meter (Accu-Check). Triglyceride levels were measured by enzymatic assay (Sigma-Aldrich), while insulin levels were quantified by ELISA (Alpco). The area under the curve of the glucose profile in response to an intraperitoneal glucose load (2 g/kg) was used for the calculation of the improvement in glucose clearance.

TABLE-US-00002 Table 1 shows the correlation between hepatic levels of expression of miR-541 and different metabolic parameters in healthy volunteers and obese subjects who are not on diabetes medications. miR-541 miR-541 vs. miR-541 vs. miR-541 miR-541 vs. miR-541 miR-541 vs. insulin HOMA-IR triglycerides vs. ASAT bilirubin vs. leptin vs. HDL r = 0.5495 r = 0.5353 r = 0.6975 r = 0.5485 r = 0.6469 r = 0.7427 r = −0.4721 p = 0.0275 p = 0.0326 p = 0.0038 p = 0.0278 p = 0.0068 p = 0.0010 ns

[0059] The invention is further characterized by the following items: [0060] 1. A composition comprising [0061] (a) an inhibitor of miR-379 or a portion or fragment thereof and an inhibitor of miR-541 or a portion or fragment thereof, and/or [0062] (b) an inhibitor of the target site of miR-379 or a portion or fragment thereof and an inhibitor of the target site of miR-541 or a portion or fragment thereof, and/or [0063] (c) a combination of an inhibitor of miR-379 or a portion or fragment thereof and an inhibitor of the target site of miR-541 or a portion or fragment thereof or a combination of an inhibitor of the target site of miR-379 or a portion or fragment thereof and an inhibitor of miR-541 or a portion or fragment thereof. [0064] 2. The composition of item 1, wherein at least one inhibitor of miR-379 or a portion or fragment thereof comprises a nucleotide sequence complementary to or hybridizing to miR-379 or a portion or fragment thereof, and at least one inhibitor of miR-541 or a portion or fragment thereof comprises a nucleotide sequence complementary to or hybridizing to miR-541 or a portion or fragment thereof. [0065] 3. The composition of item 1 or 2, wherein [0066] a portion of miR-379 has a nucleotide sequence according to SEQ ID NO: 1, wherein not more than 6 nucleotides are substituted, and [0067] a portion of miR-541 has a nucleotide sequence according to SEQ ID NO: 2, wherein not more than 8 nucleotides are substituted. [0068] 4. The composition of any one of items 1 to 3, wherein at least one inhibitor comprises a nucleic acid sequence comprising at least 10 nucleotides. [0069] 5. The composition of any one of items 1 to 4, wherein at least one inhibitor is selected from the group consisting of Tough Decoys (TuD), Decoys, antisense oligonucleotides, anti-miR, block-miR, ribozymes, external guide sequence (EGS), oligonucleotides, small interference RNA (siRNA), small temporal RNA (stRNA), short hairpin RNA (shRNA), small RNA-induced gene activation (RNAa), small activating RNA (saRNA), locked nucleic acids (LNA), antagomirs, and peptide nucleic acids (PNA). [0070] 6. The composition of item 5, wherein at least one inhibitor is a Tough Decoy RNA (TuD). [0071] 7. The composition of any one of items 1 to 6, wherein at least one inhibitor comprises a chemical modification of the nucleic acid sequence selected from the group consisting of nucleic acid analogs comprising N-acetylgalactosamine (GalNAc), phosphorothioate DNA (PS), 2′-O-methyl RNA (OMe), 2′-O-methoxy-ethyl RNA (MOE), peptide nucleic acid (PNA), N3′-P5′-phosphoroamidate (NP), 2′-fluoro-arabino nucleic acid (FANA), morpholino phosphoroamidate (MF), cyclohexene nucleic acid (CeNA), and tricycleDNA (tc-DNA). [0072] 8. The composition of any one of items 1 to 7, wherein at least one inhibitor is comprised by a delivery vehicle selected from the group consisting of adeno-associated virus (AAV), lentiviral vector, polyethylene imine (PEI), cationic liposomes, silica nanoparticles, PEGylated PLGA, and neutral lipid. [0073] 9. The composition of any one of items 1 to 8, wherein at least one inhibitor is comprised by an adeno-associated virus (AAV). [0074] 10. The composition of any one of items 1 to 9, wherein [0075] the inhibitor of miR-379 or a portion or fragment thereof comprises a nucleotide sequence of SEQ ID NO: 3, wherein not more than 5 nucleotides are substituted, and/or [0076] the inhibitor of miR-541 or a portion or fragment thereof comprises a nucleotide sequence of SEQ ID NO: 4, wherein not more than 5 nucleotides are substituted. [0077] 11. The composition of any one of items 1 to 10, comprising an inhibitor of miR-379 or a portion or fragment thereof and an inhibitor of miR-541 or a portion or fragment thereof on the same molecule, wherein said molecule comprises a nucleotide sequence complementary to or hybridizing to miR-379 or a portion or fragment thereof and a nucleotide sequence complementary to or hybridizing to miR-541 or a portion or fragment thereof. [0078] 12. The composition of item 11, wherein said inhibitor comprises the nucleotide sequence of SEQ ID NO: 5, wherein not more than 10 nucleotides are substituted. [0079] 13. The composition of any one of items 1 to 12, which is a pharmaceutical composition. [0080] 14. The composition of any one of items 1 to 13 for use in treating or preventing a metabolic disease, a disease related to a metabolic disorder, and/or cancer. [0081] 15. The composition of item 14, wherein said metabolic disease or disease related to a metabolic disorder is selected from the group consisting of glucocorticoid hormone driven metabolic dysfunction, obesity, diabetes, diabesity, metabolic syndrome, insulin resistance, hyperglycemia, (systemic) dyslipidemia, Cushing's syndrome, adverse or side effects associated with or caused by glucocorticoid (GC) treatment or excess, atherosclerosis, heart disease, stroke, (cancer) cachexia, growth defects, hepatic steatosis, NASH, and liver fibrosis.